Secondary azoospermia after a successful natural pregnancy: a primary prospective study

Background To date, there is a lack of studies conducted on males with secondary azoospermia as a potential cause of male infertility who had previously fathered children through natural conception. The current study aims to investigate the potential causes of secondary azoospermia as a presentation of male infertility as well as the prognostic factors that can impact sperm retrieval rate (SRR) while undergoing microdissection testicular sperm extraction (microTESE). Results Thirty two patients were recruited from the andrology outpatient clinic from August 2023 till January 2024. The mean age of the patients was sixty-two years old. All patients had varicoceles. Twenty seven patients (84%) had palpable varicocele grade 2 and 3 on both sides. Further multivariate logistic regression analysis of the significant factors in the univariate regression revealed that younger age (OR 0.7, 95% C.I. 0.7-1.0, p = 0.03) and having a history of coronary artery disease (CAD) were predictable factors for negative TESE outcome (OR 123.1, 95% C.I. 3.2-4748.5, P = 0.01). Conclusion It appears that the etiopathogenesis of secondary azoospermia are multifactorial. Varicocele and CAD are major factors to be considered. Future studies should be implemented deploying larger pools of patients suffering from the same condition to affirm the findings of this primary study.


Introduction
Secondary infertility is the inability to conceive after one year of regular, unprotected intercourse following term childbirth without using assisted reproductive techniques (ART) or fertility medications [1].Around one-third of cases are due to male factors [2].Male factor infertility can result from several etiologies, including varicocele, which is the cause of nearly 81% of cases of secondary male infertility [3].Aging also has a progressive impact on spermatogenesis through the cumulative deleterious effect of reactive oxygen species on spermatogenic cells together with amyloidosis in the testis [3,4].Environmental factors such as food pesticides and lifestyle factors such as smoking and eating habits affect spermatogenic cells and increase DNA fragmentation [5,6].Also, caffeinated beverages high consumption causes spermatogenic arrest and affects Sertoli cell function [5,6].There are several genetic causes of azoospermia as a primary cause of infertility, including Y chromosome microdeletions, Klinefelter, Noonan, and Kallmann syndromes, among others [7].Secondary infertility projected to 3.3 million in 2006 accounting for six out of ten infertility cases [8].Nevertheless, the topic of secondary azoospermia was rarely discussed.Thus, there is a lack of studies conducted on infertile males with secondary azoospermia who had previously fathered children through natural conception.The current study aimed to investigate the potential causes of secondary azoospermia as a presentation of male infertility as well as the prognostic factors that could impact sperm retrieval rate (SRR) while undergoing microdissection testicular sperm extraction (microTESE).

Patients and methods
Thirty two patients were recruited from the andrology outpatient clinic from August 2023 till January 2024.The institutional review board approved the work (N-265-2023) that conforms to Helsinki declaration 2013 [9].All the participants were aware about the purpose of the study and signed an informed consent prior to joining the study.

Inclusion criteria of the patients
Males complaining of secondary azoospermia after fathering children through natural conception were included in the study.

Exclusion criteria of the patients
Patients with a history of testosterone or steroid abuse, bilateral undescended testes, bilateral epididymorchitis, untreated gonorrhea, chemotherapy or obstructive azoospermia were excluded.Finally, cases of cryptozoospermia and Y chromosome microdeletion were excluded.
All patients were subjected to the following: Medical and operative histories were obtained.Patients underwent a thorough general and local examinations.Semen analysis was done according to the guidelines of the 5th edition of WHO (2010) [10].Two semen analyses were obtained on two different occasions with an abstinence period of 2 to 7 days [11].All semen analyses of the patients showed normal volume as well as normal physical characteristics.However, all of them showed absence of sperms.

Mots-clés
Azoospermie secondaire, Grossesse naturelle, microTESE surgical microscope (M320, Leica, Germany).All surgical procedures were done by an expert andrologist.Dilated tubules were identified and biopsied.Testicular tissue was examined by two senior embryologists throughout the study.If the initial search revealed the presence of sperms, the operation was terminated and the testis was closed.If the sperm search was negative, the contralateral testis was opened.Testicular tissue was obtained for histopathology that was preserved in Bouin's solution.Histopathological examination was done by an expert pathologist.Based on the morphological pattern, histopathology was classified into: hypospermatogenesis, maturation arrest, Sertoli cell only (SCO), tubular hyalinization and mixed pattern [12].
Finally, patients were divided into two groups based on the results of the microTESE and the two groups were compared based on their clinical, demographic, hormonal, and testicular histopathological findings.

Statistical analysis
The SPSS 22nd version was used to do the statistics.For the initial features of the patients, descriptive statistics were done.Univariate and multivariate logistic regression analyses and chi square tests were used to find predictors for microTESE data and histopathological diagnosis.

Results
A total of 32 patients were enrolled in our study.The mean age of the patients was sixty-two years old (Table 1).The duration of infertility ranged from one to twenty years after delivery of their youngest child (Table 1).Average sibling's age was twenty-one years old.46% of the cases smoked tobacco, 46% of the cases had diabetes mellitus, half of them had hypertension and one third had a history of coronary artery disease (CAD) (Table 1).All patients had varicoceles where 27 patients (84%) had palpable varicocele grade 2 and 3 on both sides (Table 1).The mean FSH and LH were higher than the upper normal range (15.7 mIU/mL and 10.2 IU/L, respectively) (Table 1).Total testosterone was less than 3 ng/ml in more than half of the patients (seventeen patients), with a mean below the lower normal range limit (2.5 ng/ml) (Table 1).Patients who had positive microTESE results were significantly older (mean 65.6 ± 7.1, P = 0.030), less likely to smoke (71.40% of non-smokers P = 0.004), less likely to have a history of CAD.Additionally, 85.7% of the cases had hypospermatogenesis (P < 0.001) (Table 2).
50% of patients had spermatogenic arrest or Sertoli cell only (SCO) in histopathology (Table 2).All patients who had hypospermatogenesis and SCO with spermatogenic foci were older than 50 years old (Table 2).Moreover, chi square test was performed to see if there was an association between patient's age and histopathological diagnosis and found that males older than 50 years old are more likely to give pathology results of hypospermatogenesis or SCO with spermatogenic foci (p = 0.001).Additionally, univariate logistic regression analysis was performed to see which of the patients' factors influenced the microTESE outcome.The age of the patients was inversely associated with microTESE outcome (OR 0.9, 95% C.I. 0.8-1.0,p = 0.02).Smoking (OR 11.2, 95% C.I. 1.85-68.1,p = 0.008) and a history of CAD (OR 7.2, 95% C.I. 1.3-39.6,P = 0.02) were associated with an increased risk of a negative microTESE outcome.Further

Discussion
The current study demonstrated that the included patients who previously fathered children by natural conception were currently azoospermic.Unfortunately, all included cases did not have any semen report prior to fathering their children.Furthermore, patients with positive microTESE outcome were compared to those with negative microTESE outcome regarding age, varicocele grade, diabetes milletus, CAD, smoking and hypertension.Our study had revealed that ageing and non smoking were associated with favourable microTESE outcome.In a similar trend, Li et al. ( 2018) had shown that age might have a predictive value for successful sperm recovery [13].Quite the reverse, several studies had shown that ageing as well as life style might have detrimental impact on spermatogenesis attributed to progressive damage by oxidative stress [4,6,14,15].Remarkably, there is no consensus on the cut off value for advanced paternal  [20,21].However, it should be mentioned that a critical review stressed on the fact that male age being a key determinant for the health of the offspring rather than the inability to conceive [22].Moreover, the current study had demonstrated that CAD was associated with unfavourable microTESE outcome.This finding could be explained as follow.Firstly, Haverich (2017) postulated that atherosclerosis was a microvascular disease rather than a large-vessel disease [23].Thus, testicular circulation might be negatively impacted by atherosclerosis.Secondly, ageing is a major risk factor for higher incidences of chronic disorders such as cardiovascular diseases (CVDs), neurodegenerative diseases, metabolic diseases, musculoskeletal diseases and immune-senescence diseases [24,25].Furthermore, spermidine protects against cardiac aging by enhancing left ventricular elasticity, diastolic function, and mitochondrial function [26].Additionally, It had been demonstrated that CAD, essential hypertension, and heart failure are highly impacted by spermidine levels [27].
Remarkably, a recent animal study stated that spermidine's ameliorating effect on spermatogenic disorder in the testis of mice with type 1 diabetes mellitus occurred through enhancing spermatogenic cell proliferation and activating the glycolytic pathway [28].
Another major finding of the current study was the presence of varicocele in all patients.About 84% of the included patients had clinical grade 2 and 3 varicocele.It was well established that varicocele could negatively affect endoplasmic reticulum and the unfolded protein response, which accelerated cell apoptosis [29].Furthermore, varicocele was associated with increased sperm antibodies [30], abnormal testosterone secretion and defective spermatogenesis [31] and increased cytokines mediated inflammation [32].Numerous studies had demonstrated that the SRR were notably greater in cases of hypospermatogenesis compared to mixed pathologies such as SCO syndrome and spermatogenic foci, as well as primary spermatocyte arrest [33][34][35].These findings are consistent with our own findings.Interestingly, the current study shed light on a neglected sector of infertile patients who suffered from secondary azoospermia as a cause of male infertility with the utmost importance to explore the risk factors that led to such condition through implementing future studies that deploy larger cohorts of such group of patients.

Limits of the study
Our investigation was limited by its small sample size that could be regarded as the major limitation of the current work.However, this was inevitable given the rarity of our study's targeted population.Furthermore, inability to evaluate obesity of the included cases should be seen as another limitation of the current study.Moreover, inability to do paternity test was seen as a further limitation.However, it's applicability in the current study would be questionable as it would lead to major social and interralationships instability as well as it would be rejected by the included patients.Finally, inability to evaluate the impact of the advanced paternal age represented by the current study on the health of the offspring.
However, it should be noted that there are no current precautions that can minimize the risks of advanced paternal age, nor available specific tests that can recognize individuals at increased risk [22].

Conclusion
It appears that the etiopathogenesis of secondary azoospermia are multifactorial.Varicocele and CAD are major factors to be considered.Future studies should be implemented deploying larger pools of patients suffering from the same condition to affirm the findings of this primary study.

Table 1
Shows demographic and clinical and hormonal and testicular histology characteristics of the included patients

Table 2
Shows the relationship between sociodemographic characteristics and laboratory findings and microTESE outcome among the participants N.B a primary spermatocyte arrest; b Sertoli cell only syndrome; c CAD coronary artery disease; d microTESE microsurgical testicular sperm extraction; e p value was calculated using chi square test